We plan to investigate the structural arrangement of DNA sequences in metaphase chromosomes. Specifically, we plan to define the protein-DNA interactions in the Laemmli's scaffold. The scaffold, which can be isolated by de-histonization of metaphase chromosome, is comprised of a non-histone protein complex anchored by a great many of DNA loops. Metaphase chromosomes will be isolated from synchronized chicken and human cell cultures. De-histonization of chromosomes will be performed by 2 M NaCl. The scaffold will be isolated by differential centrifugation and treated with various restriction endonucleases. The DNA remaining attached to the scaffold (attachment DNA) will be separated from the free DNA (which is located in the loops before restriction enzyme digestion) by step gradient centrifugation or by filter binding procedures. The attachment DNA will be radioactively labeled by nick-translation and annealed with cellular DNA to determine the sequence complexity. The labeled DNA will be hybridized with total polysomal poly-A containing RNA (presumably messenger RNA) to investigate if the attachment DNA fraction is enriched in structural gene sequences. Moreover, we plan to use cloned chicken genomic DNA which contains the ovalbumin gene and its flanking DNA sequences including the X and Y genes (ovalbumin-related genes) of more than 50 kb possibly 100 kb in length, to probe the sequences that attach to the scaffold. This shall offer a model system to study the specific interactions between DNA and proteins in the chromosome scaffold. A similar experimental approach will be used to investigate the scaffolding structure in interphase nuclei. We also plan to investigate the structural organization of double minutes to see if there is a scaffold structure in double minute particles. The particles have been found in metaphase cell derived from human and murine tumor origins. The proposed studies should yield some new insights into the organization of chromosome/chromatin and offer some functional aspects of chromosome physiology.